Proteases with improved enzyme stability in detergents

ABSTRACT

The present disclosure relates to proteases having an amino acid sequence with at least about 70% sequence identity to the amino acid sequence given in SEQ ID NO:2, over the entire length thereof, and comprising an amino acid substitution at at least one of the positions P9, Q62, D101, N130, G166, N187, 5216, N238 or Q271, based in each case on the numbering according to SEQ ID NO:2. The present disclosure also relates to the production and use thereof. Such proteases exhibit very good stability, in particular storage stability, while at the same time having a good cleaning performance.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. National-Stage entry under 35 U.S.C. § 371based on International Application No PCT/EP2017/055277, filed Mar. 7,2017 which was published under PCT Article 21(2) and which claimspriority to German Application No. 10 2016 204 815.5, filed Mar. 23,2016, which are all hereby incorporated in their entirety by reference.

TECHNICAL FIELD

The present disclosure lies in the field of enzyme technology. Thepresent disclosure relates to proteases from Bacillus pumilus of whichthe amino-acid sequence has been altered, in particular with respect touse in washing and cleaning agents, in order to impart improved storagestability thereto, to the nucleic acids which code for said proteases,and to the preparation of said proteases. The present disclosure furtherrelates to uses of said proteases, to methods in which said proteasesare used, and to agents, in particular washing and cleaning agents,which contain said proteases.

BACKGROUND

Proteases are among the industrially most significant enzymes of all. Inthe context of washing and cleaning agents, proteases are the longestestablished enzymes that are contained in virtually all modern,high-performance washing and cleaning agents. They cause the breakdownof protein-containing stains on the item to be cleaned. Of theseproteases, subtilisin-type proteases (subtilases, subtilopeptidases, EC3.4.21.62) are particularly significant, which proteases are serineproteases owing to the catalytically active amino acids. Said proteasesact as non-specific endopeptidases and hydrolyze any acid amide bondsthat are within peptides or proteins. Their pH optimum is usually in thehighly alkaline range. An overview of this family is found, for example,in the article “Subtilases: Subtilisin-like Proteases” by R. Siezen,pages 75-95 in “Subtilisin enzymes”, published by R. Bott and C. Betzel,New York, 1996. Subtilases are formed naturally by microorganisms. Ofthese, subtilisins that are formed by and secreted from Bacillus speciesshould be mentioned in particular as the most significant group withinthe subtilases.

Examples of the subtilisin-type proteases that are preferably used inwashing and cleaning agents are the subtilisins BPN′ and Carlsberg,protease PB92, subtilisins 147 and 309, protease from Bacillus lentus,in particular from Bacillus lentus DSM 5483, subtilisin DY, the enzymesthermitase, proteinase K and proteases TW3 and TW7, which belong to thesubtilases but no longer to the subtilisins in the narrower sense, andvariants of the mentioned proteases which have an altered amino-acidsequence by comparison with the starting protease. Proteases arealtered, selectively or randomly, by methods known from the prior art,and are thereby optimized for use in washing and cleaning agents, forexample. These methods include point, deletion or insertion mutagenesis,or fusion with other proteins or protein parts. Therefore, variants thatare appropriately optimized are known for most of the proteases knownfrom the prior art.

The European patent application EP 2 016 175 A1 discloses a proteasefrom Bacillus pumilus provided for washing and cleaning agents, forexample. In general, only selected proteases are at all suitable for usein liquid surfactant-containing preparations. In preparations of thiskind, many proteases do not demonstrate sufficient catalytic performanceor stability. In particular when used in washing agents which areusually purchased by the consumer in such an amount that several weeksor months may pass until the washing agent is finally consumed (i.e. thewashing agents need to be stored by the consumer for weeks or months),many proteases demonstrate instability, which in turn leads toinsufficient catalytic activity during the washing process. Inphosphonate-containing liquid surfactant preparations, this problem iseven more serious, for example due to the complex-forming properties ofthe phosphonates or due to disadvantageous interactions between thephosphonate and the protease.

As a result, protease- and surfactant-containing liquid formulationsfrom the prior art have the disadvantage that, after storing, saidformulations often do not have satisfactory proteolytic activity andtherefore do not demonstrate optimal cleaning performance onprotease-sensitive stains.

BRIEF SUMMARY

A protease is provided herein. The protease has an amino-acid sequencewhich has an at least about 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:2 over the entire length thereof. Theprotease has an amino acid substitution at one or more of the positionsP9, Q62, D101, N130, G166, N187, 5216, N238 and Q271, based on thenumbering according to SEQ ID NO:2.

A method for preparing a protease is also provided herein. The methodincludes substituting the amino acids at the positions which correspondto positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 in SEQ ID NO:2in a starting protease of which the sequence is at least about 70%identical to the amino acid sequence set forth in SEQ ID NO:2 over theentire length thereof.

DETAILED DESCRIPTION

The following detailed description is merely exemplary in nature and isnot intended to limit the disclosure or the application and uses of thesubject matter as described herein. Furthermore, there is no intentionto be bound by any theory presented in the preceding background or thefollowing detailed description.

It has surprisingly now been found that a subtilisin-type protease fromBacillus pumilus or a reasonably similar protease (in terms of sequenceidentity) which has an amino-acid substitution at least one of thepositions P9, Q62, D101, N130, G166, N187, 5216, N238 or Q271, in eachcase based on the numbering according to SEQ ID NO:2, is improved withregard to (storage) stability with respect to the wild type form, and istherefore particularly suitable for use in washing or cleaning agents.

In a first aspect, the present disclosure therefore relates to aprotease having an amino-acid sequence which is at least about 70%identical to the amino-acid sequence shown in SEQ ID NO:2 over theentire length thereof and has an amino-acid substitution at least one ofthe positions P9, Q62, D101, N130, G166, N187, 5216, N238 or Q271, basedin each case on the numbering according to SEQ ID NO:2.

The present disclosure also relates to a method for preparing aprotease, comprising substituting an amino acid at least one positionwhich corresponds to the position 9, 62, 101, 130, 166, 187, 216, 238 or271 in SEQ ID NO:2 in a starting protease of which the sequence is atleast about 70% identical to the amino-acid sequence shown in SEQ IDNO:2 over the entire length thereof, such that the protease has at leastone of the amino-acid substitutions P9T/S/H, Q62E, D101E, N130D, G166S,N187H, S216C, N238K or Q271E.

A “protease” within the meaning of the present patent applicationtherefore covers both the protease as such and a protease prepared usinga method as contemplated herein. All comments made with regard to theprotease therefore relate to both the protease as such and proteasesprepared using corresponding methods.

Other aspects of the present disclosure relate to the nucleic acidswhich code for these proteases, to non-human host cells which containproteases or nucleic acids as contemplated herein, to agents, inparticular washing and cleaning agents, which comprise proteases ascontemplated herein, to washing and cleaning methods, and to uses of theproteases as contemplated herein in washing or cleaning agents forremoving fat-containing stains.

“At least one”, as used herein, means one or more, i.e. one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, or more.

The present disclosure is based on the surprising finding of theinventor that an amino-acid substitution at least one of the positions9, 62, 101, 130, 166, 187, 216, 238 or 271 of the protease from Bacilluspumilus according to SEQ ID NO:2 in a protease which has an amino-acidsequence that is at least about 70% identical to the amino-acid sequenceshown in SEQ ID NO:2 such that the amino acids 9T/S/H, 62E, 101E, 130D,166S, 187H, 216C, 238K or 271E are present at the correspondingpositions brings about improved (storage) stability of this alteredprotease in washing and cleaning agents. This is particularly surprisingsince none of the above-mentioned amino-acid substitutions havepreviously been associated with increased stability of the protease.

The proteases as contemplated herein have increased stability in washingor cleaning agents, in particular when stored for about 3 or more days,about 4 or more days, about 7 or more days, about 10 or more days, about12 or more days or about 14 or more days. Proteases of this kind thatare improved in terms of performance provide for improved washingresults on proteolytically sensitive stains in a wide temperature range.

Proteases as contemplated herein have enzymatic activity, i.e. they arecapable of hydrolyzing peptides and proteins, in particular in a washingor cleaning agent. A protease as contemplated herein is therefore anenzyme which catalyzes the hydrolysis of amide/peptide bonds inprotein/peptide substrates and is therefore capable of cleaving peptidesor proteins. Furthermore, a protease as contemplated herein ispreferably a mature protease, i.e. the catalytically active moleculewithout signal peptide(s) and/or propeptide(s). Unless indicatedotherwise, the specified sequences also each relate to mature(processed) enzymes.

In various embodiments, the protease as contemplated herein contains atleast one amino-acid substitution which is selected from the groupincluding of P9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K andQ271E, based in each case on the numbering according to SEQ ID NO:2. Inmore preferred embodiments, the protease as contemplated herein containsone of the following amino-acid substitution variants: (i) P9T; (ii)Q271E; (iii) P9S and N238K; (iv) P9H; (v) N187H; (vi) S216C; (vii) Q62Eand N130D; (viii) Q62E, D101E, N130D, G166S and Q271E; or (ix) P9T,N187H, S216C and Q271E, the numbering being based in each case on thenumbering according to SEQ ID NO:2.

In a further embodiment of the present disclosure, the protease has anamino-acid sequence that is at least about 70%, about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 90.5%,about 91%, about 91.5%, about 92%, about 92.5%, about 93%, about 93.5%,about 94%, about 94.5%, about 95%, about 95.5%, about 96%, about 96.5%,about 97%, about 97.5%, about 98%, about 98.5% and about 98.8% identicalto the amino-acid sequence shown in SEQ ID NO:2 over the entire lengththereof, and has one or more of the amino acid substitutions 9T/S/H,62E, 101E, 130D, 166S, 187H, 216C, 238K or 271E at least one of thepositions 9, 62, 101, 130, 166, 187, 216, 238 or 271 in the numberingaccording to SEQ ID NO:2. In the context of the present disclosure, thefeature whereby a protease has the stated substitutions means that itcontains at least one of the corresponding amino acids at thecorresponding positions, i.e. not all of the nine positions areotherwise mutated or deleted, for example by fragmentation of theprotease. The amino-acid sequences of proteases of this kind that arepreferred as contemplated herein are shown in SEQ ID NOs 3-11.

The identity of nucleic-acid or amino-acid sequences is determined by asequence comparison. This sequence comparison is based on the BLASTalgorithm (cf. for example Altschul, S. F., Gish, W., Miller, W., Myers,E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J.Mol. Biol. 215:403-410 and Altschul, Stephan F., Thomas L. Madden,Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, andDavid J. Lipman (1997): “Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs”; Nucleic Acids Res., 25, pages3389-3402) which is established and commonly used in the prior art andis carried out, in principle, by similar series of nucleotides or aminoacids in the nucleic-acid or amino-acid sequences being assigned to oneanother. The assignment of the relevant positions shown in a table isreferred to as an “alignment”. Another algorithm available from theprior art is the FASTA algorithm. Sequence comparisons (alignments), inparticular multiple sequence comparisons, are generated using computerprograms. For example, the Clustal series (cf. for example Chenna et al.(2003): Multiple sequence alignment with the Clustal series of programs.Nucleic Acid Research 31, 3497-3500), T-Coffee (cf. for exampleNotredame et al. (2000): T-Coffee: A novel method for multiple sequencealignments. J. Mol. Biol. 302, 205-217) or programs based on theseprograms or algorithms are often used. Also possible are sequencecomparisons (alignments) using the computer program Vector NTI® Suite10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, Calif.,USA) with the specified standard parameters, the AlignX-Modul of whichprogram for the sequence comparisons is based on ClustalW. Unlessindicated otherwise, the sequence identity specified herein isdetermined using the BLAST algorithm.

A comparison of this kind makes it possible to specify the similaritybetween the compared sequences. This similarity is usually expressed inpercent identity, i.e. the percentage of identical nucleotides oramino-acid residues at the same positions or at positions thatcorrespond to one another in an alignment. In amino-acid sequences, thebroader concept of “homology” factors in conserved amino-acid exchanges,i.e. amino acids having similar chemical activity, since these usuallyhave similar chemical activities within the protein. Therefore, thesimilarity between the compared sequences can also be expressed inpercent homology or percent similarity. Information relating to identityand/or homology may apply to the entirety of the polypeptides or genesor only to individual segments. Homologous or identical segments ofdifferent nucleic-acid or amino-acid sequences are therefore defined bymatches in the sequences. Segments of this kind often have identicalfunctions. Said segments may be small and only comprise a fewnucleotides or amino acids. Segments that are this small often performfunctions that are essential to the overall activity of the protein.Therefore, it may be expedient for sequence matches to only relate toindividual, optionally small, segments. However, unless indicatedotherwise, information relating to identity or homology in the presentapplication relates to the entire length of the nucleic-acid oramino-acid sequence specified in each case.

In the context of the present disclosure, if it is stated that anamino-acid position corresponds to a numerically identified position inSEQ ID NO:2, this means that the corresponding position is assigned tothe numerically identified position in SEQ ID NO:2 in an alignment asdefined above.

In a further embodiment of the present disclosure, the protease cleaningperformance is not significantly reduced by comparison with a proteasewhich has an amino-acid sequence that corresponds to the amino-acidsequence shown in SEQ ID NO:2, i.e. said protease has at least about80%, preferably at least about 100%, more preferably at least about110%, of the reference washing performance. The cleaning performance canbe determined in a washing system which contains a washing agent in adosage of between from about 4.5 and about 7.0 grams per liter ofwashing liquor, and the protease, the proteases to be compared beingused in the same concentration (based on the active protein) and thecleaning performance with respect to a stain on cotton is determined bymeasuring the extent to which the washed textiles have been cleaned. Forexample, the washing process can be carried out for about 70 minutes ata temperature of about 40° C., and the water can have a water hardnessof between from about 15.5 and about 16.5° (German degree of hardness).The concentration of the protease in the washing agent intended for thiswashing system is from about 0.001 to about 0.1 wt. %, preferably fromabout 0.01 to about 0.06 wt. %, based on the purified active protein.

A liquid reference washing agent for a washing system of this kind canbe composed as follows (all amounts are given in percent by weight):4.4% of alkyl benzene sulfonic acid, about 5.6% of further anionicsurfactants, about 2.4% of C12-C18 Na salts of fatty acids (soaps),about 4.4% of non-ionic surfactants, about 0.2% of phosphonates, about1.4% of citric acid, about 0.95% of NaOH, about 0.01% of defoamers,about 2% of glycerol, about 0.08% of preservatives, about 1% of ethanol,and the remaining percentage of demineralized water. The dosage of theliquid washing agent is preferably between from about 4.5 and about 6.0grams per liter of washing liquor, for example about 4.7, about 4.9 orabout 5.9 grams per liter of washing liquor. Washing is preferablycarried out within a pH range of between from about pH 8 and about pH10.5, preferably between from about pH 8 and about pH 9.

Within the scope of the present disclosure, the cleaning performance isdetermined, for example, at about 40° C. using a liquid washing agent asspecified above, the washing process preferably being carried out forabout 60 minutes at about 600 rpm.

The degree of whiteness, i.e. the lightening of the stains, isdetermined using optical measurement methods, preferablyphotometrically, as a measure of cleaning performance. A device suitablefor this purpose is the spectrometer Minolta CM508d, for example. Thedevices used for the measurement are usually calibrated, in advance,against a white standard, preferably a white standard that is suppliedtherewith.

Each protease being applied in an identical manner in terms of activityensures that the relevant enzymatic properties, i.e. for examplecleaning performance on particular stains, are compared even if there issome kind of divergence in the ratio of active substance to overallprotein (the values for specific activity). In general, low specificactivity can be compensated for by adding a larger amount of protein.

Otherwise, a person skilled in the art of enzyme technology is familiarwith methods for determining protease activity, and he uses said methodsas a matter of routine. For example, methods of this kind are disclosedin Tenside, Band 7 (1970), pages 125-132. Alternatively, proteaseactivity can be determined by releasing the chromophorepara-nitroaniline (pNA) from the substratesuc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF). The protease cleavesthe substrate and releases pNA. The release of pNA causes an increase inthe extinction at about 410 nm, the temporal progression of which is ameasure of enzymatic activity (cf. Del Mar et al., 1979). Themeasurement is taken at a temperature of about 25° C., a pH of about8.6, and a wavelength of about 410 nm. The measurement time is 5 min andthe measurement interval is from about 20 s to about 60 s. Proteaseactivity is usually expressed in protease units (PU). Suitable proteaseactivities are, for example, about 2.25, about 5 or about 10 PU per mlof washing liquor. However, the protease activity is not zero.

An alternative test for establishing the proteolytic activity of theproteases as contemplated herein is an optical measurement method,preferably a photometric method. The test suitable for this purposecomprises the protease-dependent cleavage of the substrate proteincasein. Said protein is cleaved by the protease into a plurality ofsmaller subproducts. All of these subproducts have an increasedabsorption at about 290 nm by comparison with uncleaved casein, thisincreased absorption being determined using a photometer, and it thusbeing possible to draw a conclusion on the enzymatic activity of theprotease.

The protein concentration can be determined using known methods, forexample the BCA method (bicinchoninic acid;2,2′-biquinolyl-4,4′-dicarboxylic acid) or the Biuret method (A. G.Gornall, C. S. Bardawill and M. M. David, J. Biol. Chem., 177 (1948),pages 751-766). The active protein concentration can be determined, inthis respect, by titrating the active centers using a suitableirreversible inhibitor and determining the residual activity (cf. M.Bender et al., J. Am. Chem. Soc. 88, 24 (1966), pages 5890-5913).

In addition to the aforementioned amino-acid alterations, proteases ascontemplated herein can have further amino-acid alterations, inparticular amino-acid substitutions, insertions or deletions. Proteasesof this kind are developed, for example, by targeted genetic alteration,i.e. by mutagenesis methods, and optimized for particular uses or inrespect of specific properties (for example in respect of theircatalytic activity, stability, etc.). Furthermore, nucleic acids ascontemplated herein can be incorporated in recombination approaches andare thus used to produce completely new types of proteases or otherpolypeptides.

The aim is to introduce targeted mutations, such as substitutions,insertions or deletions, into known molecules, in order to improve thecleaning performance of enzymes as contemplated herein, for example. Forthis purpose, in particular the surface charges and/or isoelectric pointof the molecules, and thus their interactions with the substrate, can bealtered. For example, the net charge of the enzymes can be changed inorder to thereby influence the substrate binding, in particular for usein washing and cleaning agents. Alternatively, or in addition, thestability of the protease can be increased further still, and thus thecleaning performance thereof can be improved, by one or more appropriatemutations. Advantageous properties of individual mutations, e.g.individual substitutions, may complement one another. A protease thathas already been optimized in terms of particular properties, forexample in terms of the stability thereof during storage, can thereforealso be developed within the scope of the present disclosure.

In order to describe substitutions that affect exactly one amino-acidposition (amino-acid exchanges), the following convention is usedherein: the internationally conventional single-letter code of thenaturally present amino acid is given first, and then the associatedsequence position, and finally the amino acid that has been added. If aposition can be replaced by a number of alternative amino acids, thealternatives are separated from one another by slashes. For example,P9T/S/H means that proline can be replaced at position 9 by threonine,serine or histidine. For insertions, additional amino acids areindicated after the sequence position. For deletions, the amino acidthat has been removed is replaced with a symbol, for example a star or adash, or a Δ is put before the corresponding position. For example, P9Tdescribes the substitution of proline at position 9 by threonine, P9KTdescribes the insertion of threonine after the amino acid lysine atposition 9 and P9* or ΔP9 describes the deletion of proline at position9. This nomenclature is known to a person skilled in the art of enzymetechnology.

Therefore, the present disclosure further relates to a protease whichcan be obtained from a protease as described above acting as a startingmolecule by employing one or more conservative amino-acid substitutions,the protease still having in the numbering according to SEQ ID NO:2 atleast one of the amino-acid substitutions as contemplated herein at thepositions which correspond to positions 9, 62, 101, 130, 166, 187, 216,238 and 271 in SEQ ID NO:2, as described above. The term “conservativeamino-acid substitutions” means the exchange (substitution) of anamino-acid residue with another amino-acid residue, this exchange notresulting in a change in the polarity or charge at the position of theexchanged amino acid, e.g. the exchange of a nonpolar amino-acid residuewith another nonpolar amino-acid residue. Within the scope of thepresent disclosure, conservative amino-acid substitutions include forexample: G=A=S, I=V=L=M, D=E, N=Q, K=R, Y=F, S=T,G=A=l=V=L=M=Y=F=W=P=S=T.

Alternatively or in addition, the protease can be obtained from aprotease as contemplated herein acting as a starting molecule byemploying fragmentation, deletion, insertion or substitution mutagenesisand has an amino-acid sequence which matches that of the startingmolecule over a length of at least about 50, about 60, about 70, about80, about 90, about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 260, about 265,about 270, about 271, about 272, about 273 or about 274 interconnectedamino acids, the amino-acid substitutions contained in the startingmolecule still being present at one or more of the positions whichcorrespond to positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 inSEQ ID NO:2.

It is thus possible, for example, for individual amino acids to bedeleted from the enzyme termini or loops, without this resulting in theproteolytic activity being lost or reduced. Furthermore, by employingfragmentation, deletion, insertion or substitution mutagenesis of thiskind, the allergenicity of relevant enzymes, for example, can also bereduced and thus the usability thereof can be improved overall. Theenzymes advantageously still have their proteolytic activity even afterthe mutagenesis, i.e. the proteolytic activity thereof corresponds atleast to that of the starting enzyme, i.e. in a preferred embodiment,the proteolytic activity is at least about 80%, preferably at leastabout 90%, of the activity of the starting enzyme. Other substitutionscan also have advantageous effects. It is possible to exchange a singleamino acid or several interconnected amino acids with other amino acids.

Alternatively or additionally, said protease can be obtained as astarting molecule from a protease as contemplated herein by employingone or more conservative amino-acid substitutions, the protease havingat least one of the amino-acid substitutions P9T/S/H, Q62E, D101E,N130D, G166S, N187H, S216C, N238K or Q271E at the positions whichcorrespond to the positions 9, 62, 101, 130, 166, 187, 216, 238 and 271according to SEQ ID NO:2.

In other embodiments, said protease can be obtained as a startingmolecule from a protease as contemplated herein by employingfragmentation, deletion, insertion or substitution mutagenesis, and hasan amino-acid sequence which matches that of the starting molecule overa length of at least about 50, about 60, about 70, about 80, about 90,about 100, about 110, about 120, about 130, about 140, about 150, about160, about 170, about 180, about 190, about 200, about 210, about 220,about 230, about 240, about 250, about 260, about 265, about 270, about271, about 272, about 273 or about 274 interconnected amino acids, theprotease having at least one of the amino-acid substitutions P9T/S/H,Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positionswhich correspond to positions 9, 62, 101, 130, 166, 187, 216, 238 and271 according to SEQ ID NO:2.

In this case, the other amino-acid positions are defined by an alignmentof the amino-acid sequence of a protease as contemplated herein with theamino-acid sequence of the protease from Bacillus pumilus, as shown inSEQ ID NO:2. Furthermore, the assignment of the positions is determinedby the mature protein. In particular, this assignment is also used ifthe amino-acid sequence of a protease as contemplated herein has ahigher number of amino-acid residues than the protease from Bacilluspumilus according to SEQ ID NO:2. Proceeding from the mentionedpositions in the amino-acid sequence of the protease from Bacilluspumilus, the alteration positions in a protease as contemplated hereinare those which are precisely assigned to these positions in analignment.

Advantageous positions for sequence alterations, in particularsubstitutions, of the protease from Bacillus pumilus which whentransferred to homologous positions of the proteases as contemplatedherein are preferably of significance and impart advantageous functionalproperties to the protease are therefore the positions which correspondto positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 in SEQ ID NO:2,i.e. in the numbering according to SEQ ID NO:2, in an alignment. At thestated positions, the following amino-acid residues are present in thewild-type molecule of the protease from Bacillus pumilus: P9, Q62, D101,N130, G166, N187, 5216, N238 and Q271.

Further confirmation of the correct assignment of the amino acids to bealtered, i.e. in particular of the functional correspondence thereof,can be provided by comparison tests during which the two positionsassigned to one another on the basis of an alignment are altered in thesame way in the two proteases being compared with one another and it isobserved whether the enzymatic activity is altered in the same way inthe two proteases. If, for example, an amino-acid exchange at aparticular position of the protease from Bacillus pumilus according toSEQ ID NO:2 is associated with a change in an enzymatic parameter, forexample with the increase in the K_(M) value, and if a correspondingchange in the enzymatic parameter, thus for example also an increase inthe K_(M) value, is observed in a protease variant as contemplatedherein of which the amino-acid exchange was achieved by the same addedamino acid, this is considered to be confirmation of the correctassignment.

All elements specified can also be applied to the methods ascontemplated herein for preparing a protease. A method as contemplatedherein therefore further comprises one or more of the following methodsteps:

a) introducing one or more conservative amino-acid substitutions, theprotease having at least one of the amino-acid substitutions P9T/S/H,Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positionswhich correspond to positions 9, 62, 101, 130, 166, 187, 216, 238 and271 according to SEQ ID NO:2;b) altering the amino-acid sequence by employing fragmentation,deletion, insertion or substitution mutagenesis such that the proteasehas an amino-acid sequence which matches that of the starting moleculeover a length of at least about 50, about 60, about 70, about 80, about90, about 100, about 110, about 120, about 130, about 140, about 150,about 160, about 170, about 180, about 190, about 200, about 210, about220, about 230, about 240, about 250, about 260, about 265, about 270,about 271, about 272, about 273 or about 274 interconnected amino acids,the protease having at least one of the amino-acid substitutionsP9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at thepositions which correspond to positions 9, 62, 101, 130, 166, 187, 216,238 and 271 according to SEQ ID NO:2.

All comments made also apply to the methods as contemplated herein.

In further embodiments of the present disclosure, the protease or theprotease prepared using a method as contemplated herein is still atleast about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 90.5%, about 91%, about 91.5%, about 92%,about 92.5%, about 93%, about 93.5%, about 94%, about 94.5%, about 95%,about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%,about 98.5% and about 98.8% identical to the amino-acid sequence shownin SEQ ID NO:2 over the entire length thereof. Alternatively, theprotease or the protease prepared using a method as contemplated hereinis still at least about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 90.5%, about 91%, about 91.5%,about 92%, about 92.5%, about 93%, about 93.5%, about 94%, about 94.5%,about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%,or about 98% identical to one of the amino-acid sequences shown in SEQID NOs:3-11 over the entire length thereof. The protease or the proteaseprepared using a method as contemplated herein has an amino-acidsubstitution at least one of the positions P9, Q62, D101, N130, G166,N187, 5216, N238 and Q271, based in each case on the numbering accordingto SEQ ID NO:2. In more preferred embodiments, the amino-acidsubstitution is at least one selected from the group including ofP9T/S/H, Q62E, D101E, N130D, G1665, N187H, S216C, N238K and Q271E, basedin each case on the numbering according to SEQ ID NO:2. In morepreferred embodiments, the protease has one of the following amino-acidsubstitution variants: (i) P9T; (ii) Q271E; (iii) P9S and N238K; (iv)P9H; (v) N187H; (vi) S216C; (vii) Q62E and N130D; (viii) Q62E, D101E,N130D, G166S and Q271E; or (ix) P9T, N187H, S216C and Q271E.

The present disclosure also relates to a protease as described abovewhich is additionally stabilized, in particular by employing one or moremutations, for example substitutions, or by being coupled to a polymer.Increasing stability during storage and/or during use, for exampleduring the washing process, leads to the enzymatic activity beingmaintained for longer and thus to the cleaning performance beingimproved. In principle, all stabilizing possibilities that are expedientand/or described in the prior art can be considered for this.Stabilizations which are achieved by employing mutations of the enzymeitself are preferred, since stabilizations of this kind do not requireany further working steps after the enzyme has been obtained. Examplesof sequence alterations suitable for this purpose have been mentionedabove. Further suitable sequence alterations are known from the priorart.

Further possibilities for stabilization include for example:

-   -   altering the binding of metal ions, in particular the calcium        binding sites, for example by exchanging one or more of the        amino acid(s) that are involved in the calcium binding with one        or more negatively charged amino acids and/or by introducing        sequence alterations in at least one of the sequences of the two        amino acids arginine and glycine;    -   protecting against the influence of denaturing agents, such as        surfactants, by employing mutations which cause the amino-acid        sequence to be altered on or at the surface of the protein;    -   exchanging amino acids which are close to the N-terminus with        amino acids which are assumed to come into contact with the rest        of the molecule by employing non-covalent interactions, and thus        contribute to maintaining the globular structure.

Preferred embodiments are those in which the enzyme is stabilized inseveral different ways, since several stabilizing mutations have acumulative or synergistic effect.

The present disclosure also relates to a protease as described abovewhich has at least one chemical modification. A protease that is alteredin this way is referred to as a “derivative”, i.e. the protease isderivatized.

Within the meaning of the present application, “derivatives” aretherefore understood to mean proteins of which the pure amino-acid chainhas been modified chemically. Derivatizations of this kind can becarried out in vivo, for example, by the host cell which expresses theprotein. In this respect, couplings to low-molecular-weight compounds,such as lipids or oligosaccharides, are of particular importance.However, derivatizations may also be carried out in vitro, for exampleby the chemical conversion of a side chain of an amino acid or bycovalent bonding of another compound to the protein. For example, it ispossible to couple amines to carboxyl groups of an enzyme in order tochange the isoelectric point. This other compound may also be anotherprotein which is bound to a protein as contemplated herein viabifunctional chemical compounds, for example. Derivatization is alsounderstood to mean covalent bonding to a macromolecular carrier, ornon-covalent inclusion in suitable macromolecular cage structures.Derivatizations can, for example, influence the substrate specificity orthe bond strength to the substrate or cause temporary inhibition ofenzymatic activity, if the coupled substance is an inhibitor. This canbe expedient in terms of the period of storage, for example.Modifications of this kind can also influence stability or enzymaticactivity. They can also be used to reduce the allergenicity and/orimmunogenicity of the protein and to thus increase the skincompatibility thereof, for example. For example, couplings tomacromolecular compounds, for example polyethylene glycol, can improvethe protein in terms of stability and/or skin compatibility.

In the broadest sense, derivatives of a protein as contemplated hereincan be understood to also include preparations of these proteins.Depending on how a protein is obtained, recovered or prepared, saidprotein can be combined with a wide range of other substances, forexample from the culture of the microorganisms that produce it. Aprotein may also have been deliberately mixed with other substances inorder to increase its storage stability, for example. Therefore, thepresent disclosure also covers all preparations of a protein ascontemplated herein. This is still true irrespective of whether or notthis enzymatic activity actually develops in a particular preparation.This is because it may be desirable for the protein to not have anyactivity or to only have low activity when being stored, and for theenzymatic function to only develop once the protein is in use. This canbe controlled, for example, by appropriate accompanying substances. Inparticular, in this respect, it is possible to jointly prepare proteasesand specific inhibitors.

Among all above-described proteases or protease variants and/orderivatives, particularly preferred within the scope of the presentdisclosure are proteases, protease variants and/or derivatives of whichthe stability and/or activity corresponds to at least one of those ofthe proteases according to SEQ ID NOs: 3-11, and/or of which thecleaning performance corresponds to at least one of those of theproteases according to SEQ ID NOs: 3-11, the cleaning performance beingdetermined in a washing system, as described above.

The present disclosure also relates to a nucleic acid which codes for aprotease as contemplated herein, and to a vector containing a nucleicacid of this kind, in particular a cloning vector or an expressionvector. A nucleic acid of this kind can have the nucleotide sequenceshown in SEQ ID NO:1 as the coding sequence, which nucleotide sequenceis mutated at the corresponding positions in order to give the desiredamino-acid substitution.

These may be DNA or RNA molecules. They may be present as a singlestrand, as a single strand that is complementary to the first singlestrand, or as a double strand. In the case of DNA molecules, inparticular, the sequences of the two complementary strands should beconsidered in all three possible reading frames. It should also be notedthat different codons, i.e. base triplets, can code for the same aminoacids, such that a particular amino-acid sequence can be coded for byseveral different nucleic acids. Owing to this degeneracy of the geneticcode, all nucleic-acid sequences which can code for one of theabove-described proteases are included in this subject of the presentdisclosure. A person skilled in the art can identify these nucleic-acidsequences with absolute certainty since, despite the degeneracy of thegenetic code, defined amino acids can be assigned to individual codons.Therefore, proceeding from an amino-acid sequence, a person skilled inthe art can easily identify nucleic acids which code for said amino-acidsequence. Furthermore, in nucleic acids as contemplated herein, one ormore codons can be replaced by synonymous codons. This aspect relates inparticular to the heterologous expression of the enzymes as contemplatedherein. Therefore, each organism, for example a host cell of aproduction strain, has a particular codon usage. “Codon usage” isunderstood to mean the translation of the genetic code into amino acidsby employing the relevant organism. Bottlenecks can occur in proteinbiosynthesis if the codons on the nucleic acid are accompanied by acomparatively low number of charged tRNA molecules in the organism.Although coding for the same amino acid, this leads to a codon beingtranslated less efficiently in the organism than a synonymous codonwhich codes for the same amino acid. Owing to the presence of a highernumber of tRNA molecules for the synonymous codon, said codon can betranslated more efficiently in the organism.

Using methods which are currently generally known, such as chemicalsynthesis or the polymerase chain reaction (PCR), in conjunction withmolecular biological and/or protein chemical standard methods, it ispossible for a person skilled in the art, on the basis of known DNAand/or amino acid sequences, to produce the corresponding nucleic acidsand even complete genes. Methods of this kind are known from, forexample, Sambrook, J., Fritsch, E. F. and Maniatis, T. 2001. Molecularcloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press.

Within the meaning of the present disclosure, vectors are understood tomean elements which consist of nucleic acids and which contain a nucleicacid as contemplated herein as a characterizing nucleic-acid range.Vectors allow establishment of this nucleic acid in a species or a cellline over multiple generations or cell divisions as a stable geneticelement. Vectors are specific plasmids, i.e. circular genetic elements,in particular for use in bacteria. Within the scope of the presentdisclosure, a nucleic acid as contemplated herein is cloned in a vector.These may include vectors, for example, which originate from bacterialplasmids, from viruses, or from bacteriophages, or predominantlysynthetic vectors or plasmids having elements of various origins. Usingthe further genetic elements which are present in each case, vectors areable to become established as stable units in the host cells in questionover several generations. They may be present as separate units outsideof a chromosome or be integrated in a chromosome or chromosomal DNA.

Expression vectors have nucleic-acid sequences which enable them toreplicate in the host cells, preferably microorganisms, particularlypreferably bacteria, which contain them and to express therein acontained nucleic acid. The expression is influenced, in particular, bypromoter(s) which regulate the transcription. In principle, theexpression can be carried out by the natural promoter which isoriginally located in front of the nucleic acid to be expressed, by apromoter of the host cell provided on the expression vector, or by amodified or completely different promoter of another organism or anotherhost cell. In the present case, at least one promoter is provided forthe expression of a nucleic acid as contemplated herein and is used forthe expression thereof. Expression vectors can also be regulated, forexample by changing the culturing conditions, by reaching a particularcell density in the host cells containing said vectors, or by addingparticular substances, in particular activators for gene expression. Anexample of a substance of this kind is the galactose derivativeisopropyl-β-D-thiogalactopyranoside (IPTG) which is used as an activatorfor the bacterial lactose operon (lac operon). Unlike in expressionvectors, the contained nucleic acid in cloning vectors is not expressed.

The present disclosure also relates to a non-human host cell containinga nucleic acid as contemplated herein or a vector as contemplatedherein, or containing a protease as contemplated herein, in particular anon-human host cell which secretes the protease into the mediumsurrounding the host cell. A nucleic acid as contemplated herein or avector as contemplated herein is preferably transformed into amicroorganism which then constitutes a host cell as contemplated herein.Alternatively, individual components, i.e. nucleic-acid parts orfragments of a nucleic acid as contemplated herein, can be introducedinto a host cell such that the resulting host cell contains a nucleicacid as contemplated herein or a vector as contemplated herein. Thisprocedure is particularly suitable if the host cell already contains oneor more components of a nucleic acid as contemplated herein or of avector as contemplated herein, and the additional components are thenadded accordingly. Methods for transforming cells are established in theprior art and are sufficiently known to a person skilled in the art. Inprinciple, all cells, i.e. prokaryotic or eukaryotic cells, are suitableas host cells. Preferred host cells are those which may beadvantageously managed genetically, which involves, for example,transformation using the nucleic acid or the vector and stableestablishment thereof, for example unicellular fungi or bacteria. Inaddition, preferred host cells are distinguished by good microbiologicaland biotechnological manageability. This relates, for example, to easeof culturing, high growth rates, low demands on fermentation media, andgood production and secretion rates for foreign proteins. Preferred hostcells as contemplated herein secrete the (transgenically) expressedprotein into the medium surrounding the host cells. Furthermore, theproteases can be modified, following preparation, by the cells thatproduced them, for example by the attachment of sugar molecules, byformylations, by aminations, etc. Post-translational modifications ofthis kind can influence the protease in terms of its function.

Those host cells of which the activity can be regulated due to geneticregulation elements which are provided on the vector, for example, butwhich may also be present in these cells from the outset, representother preferred embodiments. These host cells may be induced to express,for example by the controlled addition of chemical compounds which areused as activators, by changing the culturing conditions, or uponreaching a certain cell density. This provides for cost-effectiveproduction of the proteins as contemplated herein. An example of acompound of this kind is IPTG, as described above.

Prokaryotic or bacterial cells are preferred host cells. Bacteria aredistinguished by short generation times and low demands on the culturingconditions. Cost-effective culturing methods or preparation methods canthereby be established. Furthermore, a person skilled in the art has avast pool of experience with regard to bacteria in fermentationtechnology. Gram-negative or gram-positive bacteria may be suitable forspecific production for a wide variety of reasons, which should bedetermined by experiment in any given case, for example nutrientsources, product formation rate, time constraints, etc.

In the case of gram-negative bacteria, such as Escherichia coli,numerous proteins are secreted into the periplasmatic space, i.e. thecompartment between the two membranes which enclose the cells. This maybe advantageous for particular applications. Furthermore, gram-negativebacteria may also be formed such that they secrete the expressedproteins not only into the periplasmatic space, but also into the mediumsurrounding the bacterium. By contrast, gram-positive bacteria, forexample Bacilli or actinomycetes or other representatives of theactinomycetales, have no outer membrane, and therefore secreted proteinsare released directly into the medium surrounding the bacteria,generally the nutrient medium, from which the expressed proteins may bepurified. They may be isolated directly from the medium or processedfurther. Moreover, gram-positive bacteria are related to or identical tomost origin organisms for industrially significant enzymes and theythemselves usually form comparable enzymes, such that they have asimilar codon usage and the protein synthesis apparatus thereof isnaturally aligned accordingly.

Host cells as contemplated herein may be altered in terms of theirrequirements for culture conditions, may have different or additionalselection markers, or may express different or additional proteins.These host cells may be in particular host cells that express aplurality of proteins or enzymes transgenically.

The present disclosure can be used, in principle, for allmicroorganisms, in particular for all fermentable microorganisms,particularly preferably for those from the Bacillus genus, and leads toit being possible to prepare proteins as contemplated herein by usingmicroorganisms of this kind. Microorganisms of this kind then constitutehost cells within the meaning of the present disclosure.

In a further embodiment of the present disclosure, the host cell is abacterium, preferably a bacterium selected from the group of the generaof Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium,Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, morepreferably a bacterium selected from the group of Escherichia coli,Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillusglobigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans,Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum,Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor andStenotrophomonas maltophilia.

However, the host cell may also be a eukaryotic cell which has anucleus. Therefore, the present disclosure further relates to a hostcell which has a nucleus. Unlike prokaryotic cells, eukaryotic cells areable to modify the formed protein post-translationally. Examples thereofare fungi such as actinomycetes or yeasts such as Saccharomyces orKluyveromyces. This may be particularly advantageous, for example, if,in the context of their synthesis, the proteins are intended to undergospecific modifications which systems of this kind allow. Themodifications which are carried out by eukaryotic systems, particularlyin the context of protein synthesis, include, for example, the bindingof low-molecular-weight compounds such as membrane anchors oroligosaccharides. Oligosaccharide modifications of this kind may bedesirable, for example, as a means to reduce the allergenicity of anexpressed protein. A co-expression with the enzymes formed naturally bycells of this kind, such as cellulases, can also be advantageous.Furthermore, thermophilic fungal expression systems, for example, may beparticularly suitable for expressing temperature-resistant proteins orvariants.

The host cells as contemplated herein are cultured and fermented in aconventional manner, for example in batch or continuous systems. In thefirst case, a suitable nutrient medium is inoculated with the hostcells, and the product is harvested from the medium after a period oftime that can be determined by experiment. Continuous fermentation isdistinguished by the achievement of a steady state in which, over acomparatively long period of time, some cells die, but also regenerate,and at the same time, the formed protein can be removed from the medium.

Host cells as contemplated herein are preferably used in order toprepare proteases as contemplated herein. Therefore, the presentdisclosure also relates to a method for preparing a protease,comprising:

a) culturing a host cell as contemplated herein, and

b) isolating the protease from the culture medium or from the host cell.

This subject of the present disclosure preferably includes fermentationmethods. Fermentation methods are known per se from the prior art, andconstitute the actual large-scale production step, generally followed bya suitable method for purifying the prepared product, for example theproteases as contemplated herein. All fermentation methods which arebased on a corresponding method for preparing a protease as contemplatedherein constitute embodiments of this subject of the present disclosure.

Fermentation methods which the fermentation is carried out via an inflowstrategy are in particular considered. Here, the media components thatare consumed by the continuous culturing are fed in. Significantincreases both in the cell density and in the cell mass or dry mass,and/or in particular in the activity of the protease of interest, can beachieved in this way. Furthermore, the fermentation may also be designedin such a way that undesirable metabolic products are filtered out orneutralized by adding a buffer or appropriate counterions.

The prepared protease can be harvested from the fermentation medium. Afermentation method of this kind is preferred over isolation of theprotease from the host cell, i.e. product recovery from the cell mass(dry mass); however, said method requires that suitable host cells orone or more suitable secretion markers or mechanisms and/or transportsystems be provided, so that the host cells secrete the protease intothe fermentation medium. Alternatively, without secretion, the proteasecan be isolated from the host cell, i.e. separated from the cell mass,for example by precipitation with ammonium sulfate or ethanol, or bychromatographic purification.

All aforementioned elements can be combined to form methods forpreparing proteases as contemplated herein.

The present disclosure also relates to an agent which contains aprotease as contemplated herein, as described above. The agent ispreferably a washing or cleaning agent.

This covers all conceivable types of washing or cleaning agents,including both concentrates and agents to be used in undiluted form, foruse on a commercial scale in washing machines or for washing or cleaningby hand. These agents include, for example, washing agents for textiles,carpets or natural fibers for which the term “washing agent” is used.These also include, for example, dishwashing detergents for dishwashersor manual dishwashing detergents or cleaners for hard surfaces, such asmetal, glass, porcelain, ceramics, tiles, stone, coated surfaces,plastics materials, wood or leather for which the term “cleaning agent”is used, i.e. in addition to manual and automatic dishwashingdetergents, also abrasive cleaners, glass cleaners, WC rim blocks, etc.Within the scope of the present disclosure, the washing and cleaningagents also include auxiliary washing agents, which are added to theactual washing agent when washing textiles manually or using a machinein order to achieve an additional effect. Furthermore, within the scopeof the present disclosure, washing and cleaning agents also includetextile pre-treatment and post-treatment agents, i.e. agents with whichthe piece of laundry comes into contact before it is actually washed,for example in order to loosen stubborn dirt, and also agents whichimpart other desirable properties to the laundry, for example softnessto touch, crease resistance or low static charge, in a step that comesafter the actual textile washing process. The agents mentioned lastinclude, inter alia, softeners.

The washing or cleaning agents as contemplated herein, which may bepresent in the form of powdered solids, compressed particles,homogeneous solutions or suspensions, can contain, in addition to aprotease as contemplated herein, all known ingredients that are commonin agents of this kind, at least one further ingredient preferably beingpresent in the agent. The agents as contemplated herein may inparticular contain surfactants, builders, peroxygen compounds or bleachactivators. They may also contain water-miscible organic solvents,further enzymes, sequestering agents, electrolytes, pH regulators and/orfurther auxiliaries such as optical brighteners, graying inhibitors,foam regulators, and dyes and fragrances, and combinations thereof.

In particular, a combination of a protease as contemplated herein withone or more further ingredient(s) of the agent is advantageous, since anagent of this kind has improved cleaning performance in preferredembodiments as contemplated herein on account of synergies obtainedthereby. In particular, such synergy can be achieved by the combinationof a protease as contemplated herein with a surfactant and/or a builderand/or a peroxygen compound and/or a bleach activator. Since theproteases described herein have high enzyme stability even without anenzyme stabilizer, for example boric acid, the addition of suchstabilizers to the enzyme-containing agents can be omitted or the amountof said stabilizers can be reduced in various embodiments.

Advantageous ingredients of agents as contemplated herein are disclosedin international patent application WO2009/121725, starting on thepenultimate paragraph of page 5 and ending on page 13 after the secondparagraph. Reference is made explicitly to this disclosure and thecontent thereof is incorporated in the present patent application.

An agent as contemplated herein preferably contains the protease in anamount of from about 2 μg to about 20 mg, preferably from about 5 μg toabout 17.5 mg, particularly preferably from about 20 μg to about 15 mg,and very particularly preferably from about 50 μg to about 10 mg per gof the agent. Furthermore, the protease contained in the agent and/orfurther ingredients of the agent may be encapsulated in a substance thatis impermeable to the enzyme at room temperature or in the absence ofwater, which substance becomes permeable to the enzyme under useconditions of the agent. Such an embodiment as contemplated herein theprotease is encapsulated in a substance that is impermeable to theprotease at room temperature or in the absence of water. Furthermore,the washing or cleaning agent itself can also be packaged in acontainer, preferably an airtight container, from which it is releasedshortly before use or during the washing process.

In other embodiments of the present disclosure, the agent is present insolid form, in particular as a flowable powder having a bulk density offrom about 300 g/l to about 1200 g/l, in particular from about 500 g/lto about 900 g/l, or

is present in paste or liquid form, and/or

is present in gel or pouch form, and/or

is present as a single-component system, or

is divided into a plurality of components.

These embodiments of the present disclosure cover all solid, powder,liquid, gel or paste dosage forms of agents as contemplated herein thatmay optionally also consist of a plurality of phases and may be presentin compressed or uncompressed form. The agent may be present in the formof a flowable powder, in particular having a bulk density of from about300 g/l to about 1200 g/l, more particularly from about 500 g/l to about900 g/l or from about 600 g/l to about 850 g/l. The solid dosage formsof the agent also include extrudates, granules, tablets or pouches.Alternatively, the agent may also be a liquid, gel or paste, for examplein the form of a non-aqueous liquid washing agent or a non-aqueous pasteor in the form of an aqueous liquid washing agent or water-containingpaste. Furthermore, the agent may be present as a single-componentsystem. Agents of this kind consist of one phase. Alternatively, anagent can also consist of a plurality of phases. An agent of this kindis therefore divided into a plurality of components.

Washing or cleaning agents as contemplated herein may only contain aprotease. Alternatively, they may also contain further hydrolyticenzymes or other enzymes in a concentration that is expedient in termsof the effectiveness of the agent. Another embodiment of the presentdisclosure thus relates to agents which also comprise one or morefurther enzymes. All enzymes which can develop catalytic activity in theagent as contemplated herein, in particular a lipase, amylase,cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase,xyloglucanase, β-glucosidase, pectinase, carrageenase, perhydrolase,oxidase, oxidoreductase or other proteases which are different from theprotease as contemplated herein, and mixtures thereof, can preferably beused as further enzymes. Further enzymes are contained in the agentadvantageously in an amount of from about 1×10⁻⁸ to about 5 wt. % ineach case, based on the active protein. Each further enzyme is containedin agents as contemplated herein in an amount of, in order of increasingpreference, from about 1×10⁻⁷ to about 3 wt. %, from about 0.00001 toabout 1 wt. %, from about 0.00005 to about 0.5 wt. %, from about 0.0001to about 0.1 wt. %, and most particularly preferably from about 0.0001to about 0.05 wt. %, based on the active protein. The enzymesparticularly preferably have synergistic cleaning performances withrespect to particular stains or marks, i.e. the enzymes contained in theagent composition assist one another in terms of the cleaningperformance thereof. Very particularly preferably, such synergy existsbetween the protease contained as contemplated herein and a furtherenzyme of an agent as contemplated herein, in particular between thestated protease and an amylase and/or a lipase and/or a mannanase and/ora cellulase and/or a pectinase. Synergistic effects can occur not onlybetween different enzymes, but also between one or more enzymes andother ingredients of the agent as contemplated herein.

The present disclosure further relates to a method for cleaning textilesor hard surfaces, exemplified in that an agent as contemplated herein isused in at least one method step, or in that a protease as contemplatedherein becomes catalytically active in at least one method step, inparticular such that the protease is used in an amount of from about 40μg to about 4 g, preferably from about 50 μg to about 3 g, particularlypreferably from about 100 μg to about 2 g, and very particularlypreferably from about 200 μg to about 1 g.

In various embodiments, the above-described method is distinguished inthat the protease is used at a temperature of from about 0 to about 100°C., preferably from about 0 to about 60° C., more preferably from about20 to about 40° C., and most preferably at a temperature ofapproximately 35° C.

These embodiments include both manual and automatic methods, automaticmethods being preferred. Methods for cleaning textiles are generallydistinguished in that various substances that have a cleaning effect areapplied to the item to be cleaned in a plurality of method steps andwashed off after the contact time, or in that the item to be cleaned istreated with a washing agent or a solution or dilution of this agent insome other way. The same applies to methods for cleaning all materialsother than textiles, in particular hard surfaces. All conceivablewashing or cleaning methods can be enhanced in at least one of themethod steps using a washing or cleaning agent as contemplated herein ora protease as contemplated herein, and then constitute embodiments ofthe present disclosure. All elements, subjects and embodiments that aredescribed for protease as contemplated herein and agents that containthem can also be applied to this subject of the present disclosure.Therefore, at this juncture, reference is explicitly made to thedisclosure at the corresponding point when it was indicated that thisdisclosure also applies to the above methods as contemplated herein.

Since proteases as contemplated herein naturally already have hydrolyticactivity and these also develop in media that otherwise have no cleaningforce, such as in simple buffers, an individual and/or the only step ofa method of this kind can consist in bringing a protease as contemplatedherein into contact with the stain as the only component that has acleaning effect, preferably in a buffer solution or in water. Thisconstitutes a further embodiment of this subject of the presentdisclosure.

Methods for treating textile raw materials or for textile care in whicha protease as contemplated herein becomes active in at least one methodstep also constitute alternative embodiments of this subject of thepresent disclosure. Of such methods, methods for textile raw materials,fibers or textiles having natural components are preferred, and veryparticularly for those containing wool or silk.

Finally, the present disclosure further relates to the use of theproteases described herein in washing or cleaning agents, for example asdescribed above, for (improved) removal of fat-containing stains, forexample from textiles or hard surfaces.

All elements, subjects and embodiments that are described for proteaseas contemplated herein and agents that contain them can also be appliedto this subject of the present disclosure. Therefore, at this juncture,reference is explicitly made to the disclosure at the correspondingpoint when it was indicated that this disclosure also applies to theabove use as contemplated herein.

EXAMPLES

All working steps involving molecular biology followed standard methods,as specified, for example, in the manual by Fritsch, Sambrook andManiatis “Molecular cloning: a laboratory manual”, Cold Spring HarbourLaboratory Press, New York, 1989, or comparable relevant pieces of work.Enzymes and kits were used according to the manufacturer's instructionsin each case.

Overview of the Mutations

SEQ Variant Sequence ID NO: Variant 1 P9T 3 Variant 2 Q271E 4 Variant 3P9S N238K 5 Variant 4 P9H 6 Variant 5 N187H 7 Variant 6 S216C 8 Variant7 Q62E N130D 9 Variant 8 Q62E D101E N130D G166S Q271E 10 Variant 9 P9TN187H S216C Q271E 11

Example 1: Storage Stability of the Enzyme in Washing Agent

The proteases are stirred in a washing agent matrix at the same activitylevel and stored at 30° C. Using a standard activity assay for proteases(hydrolysis of suc-AAPF-pNA), the initial activity and the remainingactivity of the protease is measured after 42 hours and 9 days ofstorage at 30° C.

In order to generate harsh conditions, the proteases are stored once inthe washing agent matrix without a stabilizer (boric acid) and, forcomparison, once with boric acid.

This is the washing agent matrix that has been used in this case(LSPA+):

Wt. % active Wt. % active substance in the substance in the Chemicalname raw material formulation demineralized water 100 remainderalkylbenzene sulfonic acid 96 4.4 anionic surfactants 70 5.6 C12-C18fatty acid Na salt 30 2.4 non-ionic surfactants 100 4.4 phosphonates 400.2 citric acid 100 1.4 NaOH 50 0.95 defoamers t.q. 0.01 glycerol 100 2preservatives 100 0.08 ethanol 93 1 without optical brighteners,perfume, dye and enzymes.This matrix is provided for the measurements using stabilizer with 1%boric acid.

Example 2: Enzymes

The proteases are present in Bacillus subtilis in supernatants generatedin shake flasks. Said proteases are diluted to the same activity level.50% washing agent matrix+/−boric acid are mixed with 50% correspondinglydiluted Bacillus subtilis protease supernatant and mixed well. Theclosed glasses are incubated at 30° C. At the time the sample is taken,a certain amount of matrix/protease mixture is removed and dissolved bystirring for 20 minutes at RT in the sample buffer (0.1 M Tris/HCl, pH8.6). The AAPF assay is then carried out as described below.

9 mutants (SEQ ID NOs 3-11) have proven to be advantageous; the activityis shown in % of the initial value. This was measured immediately afterintermixing the proteases with the matrix and is set at 100% in eachcase:

Initial 4 d − 4 d + 12 d − 12 d + Protease activity B(OH)₃ B(OH)₃ B(OH)₃B(OH)₃ SEQ ID NO: 2 100% 78% 106% 38% 88% SEQ ID NO: 3 100% 99% 103% 77%92% SEQ ID NO: 4 100% 91% 103% 56% 94% SEQ ID NO: 5 100% 98% 105% 83%90% SEQ ID NO: 6 100% 102%  100% 82% 92% SEQ ID NO: 7 100% 96% 111% 63%95% SEQ ID NO: 8 100% 99% 106% 73% 97%

The mutants according to SEQ ID NOs 9 to 11 are recombinants which havebeen measured in another test:

Initial 3.5 d − 3.5 d + Protease activity B(OH)₃ B(OH)₃ SEQ ID NO: 2100% 76%  98% SEQ ID NO: 9 100% 93% 110% SEQ ID NO: 10 100% 88% 112% SEQID NO: 11 100% 93% 101%

All mutants demonstrate a washing performance comparable to that of thewild type, i.e. the washing performance thereof is at most 10% poorer,which is within the limits of measurement variation (results not shown).

Example 3: Protease Activity Assays

The activity of the proteases is determined by releasing the chromophorepara-nitroaniline from the substrate succinylalanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; BachemL-1400). The release of pNA causes an increase in the extinction at 410nm, the temporal progression of which is a measure of enzymaticactivity.

The measurement is taken at a temperature of 25° C., a pH of 8.6, and awavelength of 410 nm. The measurement time was 5 minutes at ameasurement interval of 20 to 60 seconds.

Measurement Formulation

10 μL AAPF solution (70 mg/mL)

1000 μL Tris/HCl (0.1 M; pH 8.6 with 0.1% Brij 35)

10 μL diluted protease solution

Kinetics produced over 5 minutes at 25° C. (410 nm)

The invention claimed is:
 1. A protease having an amino-acid sequencewhich has an at least 90% sequence identity to the amino acid sequenceset forth in SEQ ID NO:2 over the entire length thereof and which has anamino acid substitution at one or more of the positions P9, Q62, D101,N130, G166, N187, S216, N238 and Q271, each based on the numberingaccording to SEQ ID NO:2.
 2. The protease according to claim 1, whereinthe amino acid substitution is selected from the group consisting ofP9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K Q271E, andcombinations thereof, each based on the numbering according to SEQ IDNO:2.
 3. The protease according to claim 1, wherein the protease has oneof the following amino acid substitution variants, each based on thenumbering according to SEQ ID NO:2: (i) P9T; (ii) Q271E; (iii) P9S andN238K; (iv) P9H; (v) N187H; (vi) S216C; (vii) Q62E and N130D; (viii)Q62E, D101E, N130D, G166S and Q271E; or (ix) P9T, N187H, S216C andQ271E.
 4. A protease wherein (a) said protease can be obtained as astarting molecule from a protease according to claim 1 by employing oneor more conservative amino acid substitutions, the protease having atleast one of the amino acid substitutions P9T/S/H, Q62E, D101E, N130D,G166S, N187H, S216C, N238K or Q271E at the positions which correspond tothe positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 according toSEQ ID NO:2; and/or (b) said protease can be obtained as a startingmolecule from a protease according to claim 1 by employingfragmentation, deletion, insertion or substitution mutagenesis, and hasan amino acid sequence which matches that of the starting molecule overa length of at least about 50 interconnected amino acids, the proteasehaving at least one of the amino acid substitutions P9T/S/H, Q62E,D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positions whichcorrespond to positions 9, 62, 101, 130, 166, 187, 216, 238 and 271according to SEQ ID NO:2.
 5. A method for preparing a protease,comprising substituting an amino acid at one or more of the positionswhich correspond to positions 9, 62, 101, 130, 166, 187, 216, 238 and271 in SEQ ID NO:2 in a starting protease of which the sequence is atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:2over the entire length thereof.
 6. The method according to claim 5,further comprising one or both of the following method steps: (a)introducing one or more conservative amino-acid substitutions, whereinthe protease has at least one of the amino acid substitutions P9T/S/H,Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positionswhich correspond to the positions 9, 62, 101, 130, 166, 187, 216, 238and 271 according to SEQ ID NO:2; (b) altering the amino-acid sequenceby employing fragmentation, deletion, insertion or substitutionmutagenesis such that the protease has an amino acid sequence whichmatches that of the starting molecule over a length of at least about 50interconnected amino acids, wherein the protease has at least one of theamino acid substitutions P9T/S/H, Q62E, D101E, N130D, G166S, N187H,S216C, N238K or Q271E at the positions which correspond to positions 9,62, 101, 130, 166, 187, 216, 238 and 271 according to SEQ ID NO:2. 7.The A nucleic acid coding for the protease according to claim
 1. 8. Avector comprising the nucleic acid according to claim
 7. 9. A non-humanhost cell comprising the vector according to claim
 8. 10. A cleaningagent comprising the protease according to claim 1 and a surfactant. 11.A method of cleaning textiles or a hard surface, comprising contactingthe textiles or hard surface with a cleaning agent according to claim10.
 12. The protease according to claim 1, wherein the protease has theamino acid substitution P9T based on the numbering according to SEQ IDNO:2.
 13. The protease according to claim 1, wherein the protease hasthe amino acid substitution Q271E based on the numbering according toSEQ ID NO:2.
 14. The protease according to claim 1, wherein the proteasehas the amino acid substitutions P9S and N238K based on the numberingaccording to SEQ ID NO:2.
 15. The protease according to claim 1, whereinthe protease has the amino acid substitution P9H based on the numberingaccording to SEQ ID NO:2.
 16. The protease according to claim 1, whereinthe protease has the amino acid substitution N187H based on thenumbering according to SEQ ID NO:2.
 17. The protease according to claim1, wherein the protease has the amino acid substitution S216C based onthe numbering according to SEQ ID NO:2.
 18. The protease according toclaim 1, wherein the protease has the amino acid substitutions Q62E andN130D based on the numbering according to SEQ ID NO:2.
 19. The proteaseaccording to claim 1, wherein the protease has the amino acidsubstitutions Q62E, D101E, N130D, G166S and Q271E based on the numberingaccording to SEQ ID NO:2.
 20. The protease according to claim 1, whereinthe protease has the amino acid substitutions 9T, N187H, S216C and Q271Ebased on the numbering according to SEQ ID NO:2.